| In Chinese Medicine (CM), ischaemic stroke belongs to the category of "collateral diseases", its pathogenesis is based on "toxic brain meridian damage". Chinese herbal formula "TongluoJiu’nao injection fluid", which is developed on the basis of relieving "toxic brain meridian damage", is a pure injective herbal medical fluid. Using the treatment method of detoxification and unclogging the meridians as the basis, selecting Gardenia Cape Jasmineas the main herb.The haemo stasic, vessel-cleaning and stasis-circulating characteristics of Panaxpseudo Ginseng as side herb, both of which are compatible and detoxifies, calming the blood and unclogging the meridians,thus playing abeneficialrole inprotectingthe brain during is chaemic damage to the brain. Early results show:TongluoJiu’naoinjection fluid possesses the beneficial effects of amongst others reducing is chaemic brain oedema, reduceinfarctsize, and improve nervecell damage. Clinical trialsalso showed that TongluoJiu’naoinjection fluid can significantly improve the clinical symptoms of stroke patients and reduce neurological deficits.This articleprovides experimental evidence for the study of modern Chinese Medicinal recipes by studying in-depth the effects of TongluoJiu’naoinjection fluid and its effective components of Geniposide and PanaxNotoginsengSaponins (PNS)in oxidative damage of microvascular endothelial cells and vascular growth factor expression mechanisms. This in-depthstudy of the efficacy and mechanism of drug matching enriches the biological meaning of detoxification and unclogging meridians", also providing a preliminary discussion of the compatibility features of Chinese Medicine.Objective:In this experiment,cerebral microvascular endothelial cellsare taken as the starting point. A model of ischaemic damage due to oxygen and glucose deprivation was prepared. From the facets of resisting oxidative damage and expression of vascular growth factors, we observe the pharmacodynamic and endothelial-protective characteristics of TongluoJiu’nao injection fluid and the two effective components Geniposide and PNS, from the molecular level to the scientific interpretation of the pharmaco dynamic mechanisms of drug efficacies, thus providing experimental evidencefor the scientific component behind compatibility features of Chinese Medicine. Mothods:1.Using ubiquitous methods to obtain rat brain microvascular endothelial cells, culturing, purifying and reproducing them. Using the method of oxygen-glucose deprivation to emulate cell ischaemic injury.2.Selecting the drug concentrations of TongluoJiu’nao injection fluid and effective component Geniposide、 PNS:observe the reaction of differing concentrations of three drugs on the activity of normal and ischaemic-damaged Microvascular Endothelial Cells (MVEC), thus determining the efficacy of the three drugs and their optimal concentration, using CCK-8method to detect cell activity.3.Observing TongluoJiu’nao injection fluid, and effective component Geniposide> PNS activity on MVEC with ischaemic injury;using CCK-8method to detect cell activity.4.Observing TongluoJiu’nao injection fluid and effective component Geniposide, PNS effects on the oxidative sensitivity of ischaemically-injured MVEC;using biochemical sensing to detectchanges to the malonicdialdehyde (MDA) content and superoxidase dismutase (SOD) activity of both normal and ischaemically-damaged MVEC due to the three drugs.5.Observing effects of TongluoJiu’nao injection fluid and effective component Geniposide、PNS on the expression of vascular endothelial growth factor (VEGF), brain derived nerve factor (BDNF) in ischaemically-damaged MVEC;Using irnmunohistochemical Western blot method and immunofluorescence assay to detect endothelial expression of VEGF, BDNF in normal and ischaemically-damaged MVEC.Results:1.Determination of optimal drug concentrations of three drugs using the CCK-8method:TongluoJiu’nao injection fluid4μl/ml, Geniposide33.2μg/ml, and22μg/ml of PNS are the optimal drug concentrations.2.Effects of three drugs on the activity of ischaemically-damaged MVEC:three drugs could increase the activity of endothelial cells exposed to ischaemicdamage, and all have significantly different effects. Compared with the PNS group, cell activity in the TongluoJiu’nao group increased more significantly (P<0.05).3.Effects of three drugs on the oxidative sensitivity of ischaemically-damaged MVEC: after depriving the cerebral microvascular endothelial cells of oxygen and glucose, MDA content increased significantly and SOD activity decreased significantly, indicating that during cerebral ischaemia, enhanced oxidative stress damage cells. Geniposide can effectively reduce the content of MDA in damaged cells, in this aspect, the effect of PNS is not obvious. However, PNS can significantly increase the SOD activity after ischaemicdamage, in this aspect, the effect of Geniposideis not obvious.4.Effects of the three drugs on the expression of endothelial growth factors VEGF and BDNF within ischaemically-damaged MVEC:immunohistochemical Western blot results show:Significantly increased expression of VEGF in the three treatment groups compared with the control group, PNS and TongluoJiu’nao groupsshow significantly higher BDNF expressions than that of the control group, in the geniposide group this is not obvious. Immunofluorescence results are consistent with the immunohistochemical Western blot method.Conclusion:1.TongluoJiu’naoinjectionfluid and effective components Geniposide and PNS can be effective in reducing ischaemic damage to MVEC due to oxidative stress, therefore improving cell activity. The antioxidative characteristics of Geniposide could be linked with the inhibition of free radical production and lipid peroxidation, PNS could be responsible for removing free radicals. In this regard, the two components work compatibly, complementary and synergistically in order to fight the effects of ischaemic damage on the endothelial cells. Both components not only’collaborate’ with each other, they also show’synergizing’ effects, thus showing a therapeutic advantage of integrative Traditional Chinese Medicine.2. TongluoJiu’nao injection fluid and the effective components Geniposide and PNS could, through positive regulation of the expressions of endothelial growth factors VEGF and BDNF, promote new growth of endothelial cells after ischaemic damage, recovery of blood supply to the areas inflicted with ischaemic damage, improving conditions to promote microvascular regeneration. PNS plays a major role, in both groups it improved the blood supply of oxygen into the ischaemic area, and protectsischaemic brain tissue.In conclusion, Geniposide play vascular protective effect may be through its inhibition of free radical production and lipid peroxidation, which may be related Gardenia "cooling blood detoxification".PNS may play a role in protecting blood vessels by increasing their free radical Clear and increasing vascular growth factor VEGF、BDNF expression which may be related to PNS" tonic,meridians "effect.Both collaborative compatibility, played "JieDuTongLuo" effect, showing the Chinese Trad itional Medicine Combination therapy play integration advantages regulation. |
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